Mitochondria are central to the physiology of all eukaryotic cells. The immense diversity of mitochondria and their functions among the various branches of eukaryotic organisms is likely to have evolved in response to the diverse environmental niches of these organisms, which dictate the physiological demands placed on their mitochondria. The mitochondrion of malaria parasites has characteristics that are highly divergent from their hosts'. In the 1980s, our laboratory discovered the mitochondrial DNA (mtDNA) of malaria parasites. With its highly diminished gene content and organization, this genome presented the specter of divergent mitochondrial functions in Apicomplexan parasites that could be targets for novel antimalarial drugs. Previous studies from our laboratory have validated the parasite mitochondrion as a target for antimalarial drugs. The availability of genomic sequences and advances in gene transfer technology for malaria parasites has permitted us to explore various nuclearly encoded mitochondrial functions to assess their role in parasite physiology. Findings from this project have successfully addressed questions of long standing regarding the roles of major mitochondrial metabolic functions in P. falciparum: mitochondrial electron transport chain (mtETC), tricarboxylic acid (TCA) cycle, and heme biosynthesis. For the next funding period of this project, we wish to explore additional metabolic features of the parasite mitochondria that are essential for parasite survival and might be divergent from those in their mammalian counterparts. We have initiated experiments to derive a proteomic landscape of the parasite mitochondrion at different lifecycle stages. While RNAi strategy is not feasible for P. falciparum, several advances in approaches for genetic manipulation of P. falciparum, such as CRISPR-Cas9 editing and conditional knockdown of gene expression, have become available during the last year (and are currently used successfully in our laboratory), which would permit relatively rapid gene knock-in/knockout as well as conditional knockdown mutant generation involving critical metabolic pathways. Phenotypic characterization of these mutant parasites using various methods including metabolomics would bring our understanding of the unusual mitochondrial physiology of malaria parasite to an unprecedented level, which could inform future discoveries to control malaria. We will derive an enhanced proteomic view of P. falciparum mitochondrion through the use of proximity biotinylation and allied approaches. We will investigate functions of mitochondrial transport proteins through genetic manipulation and phenotypic characterization by employing newly developed robust conditional gene knockdown approaches and gene editing. In addition, we will study components of the mtETC and the ATP synthase complex to understand their functions through the use genetic manipulations that would either disable their enzymatic functions and/or through conditional knockdown of their expression.